Single cell RNA-Seq of the murine non-myocyte cardiac cellulome
ABSTRACT: We characterized single-cell transcriptional profiles of the cardiac non-myocyte cell pool in C57BL/6J mice. The cell preparation we sequenced consisted of metabolically active, nucleated non-myocyte cells from heart ventricles of female and male mice which were depleted of endothelial cells. The goals of this experiment included examining cellular diversity, identifying markers of understudied cell populations, exploring functional roles of different cell types, and characterizing sexual dimorphism in cardiac gene expression.
Project description:Rationale: Neonatal mice have the capacity to regenerate their hearts in response to injury, but this potential is lost after the first week of life. The transcriptional changes that underpin mammalian cardiac regeneration have not been fully characterized at the molecular level. Objective: The objectives of our study were to determine if myocytes revert the transcriptional phenotype to a less differentiated state during regeneration and to systematically interrogate the transcriptional data to identify and validate potential regulators of this process. Methods and Results: We derived a core transcriptional signature of injury-induced cardiac myocyte regeneration in mouse by comparing global transcriptional programs in a dynamic model of in vitro and in vivo cardiac myocyte differentiation, in vitro cardiac myocyte explant model, as well as a neonatal heart resection model. The regenerating mouse heart revealed a transcriptional reversion of cardiac myocyte differentiation processes including reactivation of latent developmental programs similar to those observed during de-stabilization of a mature cardiac myocyte phenotype in the explant model. We identified potential upstream regulators of the core network, including interleukin 13 (IL13), which induced cardiac myocyte cell cycle entry and STAT6/STAT3 signaling in vitro. We demonstrate that STAT3/periostin and STAT6 signaling are critical mediators of IL13 signaling in cardiac myocytes. These downstream signaling molecules are also modulated in the regenerating mouse heart. Conclusions: Our work reveals new insights into the transcriptional regulation of mammalian cardiac regeneration and provides the founding circuitry for identifying potential regulators for stimulating heart regeneration. Comparison of transcriptional programs of primary myocardial tissues sampled from neonatal mice and murine hearts undergoing post-injury regeneration, along with in vitro ESC-differentiated cardiomyocytes
Project description:Myocyte enhancer factor 2 contributes as a transcription factor to cardiac remodeling processes. We wanted to link and compare known myocyte enhancer factor 2 targets to overall targets. Therefore, we used a model of neonatal rat cardiomyocytes and precipitated sheared chromatin samples with 5µg of MEF2 Ab.
Project description:Barcode swapping results in the mislabeling of sequencing reads between multiplexed samples on the new patterned flow cell Illumina sequencing machines. This may compromise the validity of numerous genomic assays, especially for single-cell studies where many samples are routinely multiplexed together. The severity and consequences of barcode swapping for single-cell transcriptomic studies remain poorly understood. We have used two statistical approaches to robustly quantify the fraction of swapped reads in each of two plate-based single-cell RNA sequencing datasets. We found that approximately 2.5% of reads were mislabeled between samples on the HiSeq 4000 machine, which is lower than previous reports. We observed no correlation between the swapped fraction of reads and the concentration of free barcode across plates. Further- more, we have demonstrated that barcode swapping may generate complex but artefactual cell libraries in droplet-based single-cell RNA sequencing studies. To eliminate these artefacts, we have developed an algorithm to exclude individual molecules that have swapped between samples in 10X Genomics experiments, exploiting the combinatorial complexity present in the data. This permits the continued use of cutting-edge sequencing machines for droplet-based experiments while avoiding the confounding effects of barcode swapping. This data repository contains the sequencing files associated with the droplet based scRNA-seq dataset in Griffiths et al. (2018). The data presented here should purely used for technical analysis, the biological motivation is nonetheless briefly described in the following: The mammary gland is a unique organ as it undergoes most of its development during puberty and adulthood. Characterising the hierarchy of the various mammary epithelial cells and how they are regulated in response to gestation, lactation and involution is important for understanding how breast cancer develops. Recent studies have used numerous markers to enrich, isolate and characterise the different epithelial cell compartments within the adult mammary gland. However, in all of these studies only a handful of markers were used to define and trace cell populations. Therefore, there is a need for an unbiased and comprehensive description of mammary epithelial cells within the gland at different developmental stages. To this end we used single cell RNA sequencing (scRNAseq) to determine the gene expression profile of individual mammary epithelial cells across four adult developmental stages; nulliparous, mid gestation, lactation and post weaning (full natural involution).
Project description:Differentiation into diverse cell lineages requires orchestration of gene regulatory networks guiding cell fate choices. Genetic factors acting through changes in transcriptional levels can contribute to cardiovascular disease risk by impacting early stages of development and have cell type-specific effects. We set out to characterize lineage trajectory progression of subpopulations and identify potential disease-related genes by examining their expression changes in single cells during early stages of cardiac lineage specification. Using 43,168 single-cell transcriptomes, we developed novel classification and trajectory analysis methods to dissect cellular composition and gene networks across five discrete time points underlying lineage derivation of mesoderm, definitive endoderm, vascular endothelium, cardiac precursors, and definitive cell types that comprise cardiomyocytes and a previously unrecognized cardiac outflow tract population.
Project description:We used human embryonic stem cell-derived retinal ganglion cells (RGCs) to characterize the transcriptome of 1,174 cells at the single cell level. The human embryonic stem cell line BRN3B-mCherry A81-H7 was differentiated to RGCs using a guided differentiation approach. Cells were harvested at day 36 and incubated with THY1 antibody (Miltenyi) before undergoing FACS. THY1 positive and THY1 negative cells were subsequently prepared for single cell RNA sequencing. Single cell suspensions were loaded onto 10X Genomics Single Cell 3' Chips along with the reverse transcription master mix as per the manufacturer's protocol for the Chromium Single Cell 3' v2 Library (10X Genomics; PN-120233), to generate single cell gel beads in emulsion. Libraries were then sequenced on an Illumina HiSeq 2500.
Project description:We identified 112 known genes and 150 expressed sequence tags (ESTs) showing more than a 1.3 fold change in target-exposed neurons compared to neurons grown in the absence of target. Expression of 36 genes and 49 ESTs was upregulated, while expression of 76 genes and 101 ESTs was downregulated by the presence of target during the culture period. Overall, these changes represented approximately 1.6% of the probe set. Keywords: Patterns of gene expression by neuron-target interaction Overall design: We investigated the molecular basis of the target-regulated growth inhibition program using DNA microarrays to analyze patterns of gene expression following contact of sympathetic neurons with their myocyte targets. Total RNA was isolated from sympathetic neurons cultured alone and from neurons isolated from myocyte co-cultures. The two neuron populations showed minimal levels of atrial natriutic peptide (ANF) mRNA, a myocyte marker, compared to myocyte-containing cultures, indicating a low level of myocyte contamination for neurons purified from co-cultures. Isolated total RNA was reverse transcribed, labeled with biotin by in vitro transcription and hybridized to an Affymetrix Rat Expression Set 230A microarray with 15,900 probe sets. The hybridized microarray was washed, stained, scanned and quantified using DNA-Chip Analyzer (dChip) 1.3 (Li and Wong, 2001).
Project description:We identified 112 known genes and 150 expressed sequence tags (ESTs) showing more than a 1.3 fold change in target-exposed neurons compared to neurons grown in the absence of target. Expression of 36 genes and 49 ESTs was upregulated, while expression of 76 genes and 101 ESTs was downregulated by the presence of target during the culture period. Overall, these changes represented approximately 1.6% of the probe set. Experiment Overall Design: We investigated the molecular basis of the target-regulated growth inhibition program using DNA microarrays to analyze patterns of gene expression following contact of sympathetic neurons with their myocyte targets. Total RNA was isolated from sympathetic neurons cultured alone and from neurons isolated from myocyte co-cultures. The two neuron populations showed minimal levels of atrial natriutic peptide (ANF) mRNA, a myocyte marker, compared to myocyte-containing cultures, indicating a low level of myocyte contamination for neurons purified from co-cultures. Experiment Overall Design: Isolated total RNA was reverse transcribed, labeled with biotin by in vitro transcription and hybridized to an Affymetrix Rat Expression Set 230A microarray with 15,900 probe sets. The hybridized microarray was washed, stained, scanned and quantified using DNA-Chip Analyzer (dChip) 1.3 (Li and Wong, 2001).
Project description:TLDA miRNA profiling on purified rat cardiomyocytes (Myo) (Ctl) and myocyte-derived progenitor cells (MDCs) demonstrated significant dedifferentiation of myocytes and identity of stemness, cell cycle progression and proliferation in MDCs after continuous culture in mitogen-rich medium for about 2 weeks. Total RNA was extracted from 3 batches of myocytes or MDCs; 100ug each was subjected to TLDA microRNA profiling after preamplification using ABI's kit. Cardiomyocyte (Myo, Ctl) was used as calibrator sample.
Project description:This SBML representation of the Homo sapiens myocyte metabolic network is made available under the Creative Commons Attribution-Share Alike 3.0 Unported Licence (see www.creativecommons.org).